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An abstract of the conference paper "Replication of the association of the COG6 gene with juvenile idiopathic arthritis (JIA)," by Femke Van Wijk and colleagues is presented.

Background: Juvenile idiopathic arthritis is the most common chronic rheumatic disease of childhood. The term JIA encompasses a heterogenous group of diseases. The variability in phenotype of patients affected by the disease means it is not uncommon for mimics of JIA to be misdiagnosed., Case Presentation: We present four cases who were treated in single tertiary rheumatology centre for JIA who were subsequently diagnosed with a rare monogenic disease. All four patients shared the unifying features of presenting in early childhood and subsequently suffered with refractory disease, not amenable to usual standards of treatment. Multicentric Carpotarsal Osteolysis Syndrome and Camptodactyly-arthropathy-coxa vara-pericarditis syndrome are non-inflammatory conditions and patients typically present with arthropathy, normal inflammatory markers and atypical radiological features. Blau syndrome is an autosomal dominant condition and patients will typically have symmetrical joint involvement with a strong family history of arthritis, signifying the genetic aetiology., Conclusions: We share our learning from these cases to add to the growing portfolio of JIA mimics and to highlight when to consider an alternative diagnosis. In cases of refractory disease and diagnostic uncertainty further imaging and genetic testing can play a crucial role in establishing the aetiology. In all of these cases the correct diagnosis was made due to careful, longitudinal clinical phenotyping and a close working relationship between rheumatology, radiology and clinical genetics; highlighting the importance of the multidisciplinary team in managing complex patients., (© 2022. The Author(s).)

Background: Genome-wide association studies (GWAS) have uncovered many genetic risk loci for psoriasis, yet many remain uncharacterised in terms of the causal gene and their biological mechanism in disease. This is largely a result of the findings that over 90% of GWAS variants map outside of protein-coding DNA and instead are enriched in cell type- and stimulation-specific gene regulatory regions., Results: Here, we use a disease-focused Capture Hi-C (CHi-C) experiment to link psoriasis-associated variants with their target genes in psoriasis-relevant cell lines (HaCaT keratinocytes and My-La CD8+ T cells). We confirm previously assigned genes, suggest novel candidates and provide evidence for complexity at psoriasis GWAS loci. For one locus, uniquely, we combine further epigenomic evidence to demonstrate how a psoriasis-associated region forms a functional interaction with the distant (> 500 kb) KLF4 gene. This interaction occurs between the gene and active enhancers in HaCaT cells, but not in My-La cells. We go on to investigate this long-distance interaction further with Cas9 fusion protein-mediated chromatin modification (CRISPR activation) coupled with RNA-seq, demonstrating how activation of the psoriasis-associated enhancer upregulates KLF4 and its downstream targets, relevant to skin cells and apoptosis., Conclusions: This approach utilises multiple functional genomic techniques to follow up GWAS-associated variants implicating relevant cell types and causal genes in each locus; these are vital next steps for the translation of genetic findings into clinical benefit.

Background: The composition of the skin microbiome is predicted to play a role in the development of conditions such as atopic eczema and psoriasis. 16S rRNA gene sequencing allows the investigation of bacterial microbiota. A significant challenge in this field is development of cost effective high throughput methodologies for the robust interrogation of the skin microbiota, where biomass is low. Here we describe validation of methodologies for 16S rRNA (ribosomal ribonucleic acid) gene sequencing from the skin microbiome, using the Illumina MiSeq platform, the selection of primer to amplify regions for sequencing and we compare results with the current standard protocols.., Methods: DNA was obtained from two low density mock communities of 11 diverse bacterial strains (with and without human DNA supplementation) and from swabs taken from the skin of healthy volunteers. This was amplified using primer pairs covering hypervariable regions of the 16S rRNA gene: primers 63F and 519R (V1-V3); and 347F and 803R (V3-V4). The resultant libraries were indexed for the MiSeq and Roche454 and sequenced. Both data sets were denoised, cleaned of chimeras and analysed using QIIME., Results: There was no significant difference in the diversity indices at the phylum and the genus level observed between the platforms. The capture of diversity using the low density mock community samples demonstrated that the primer pair spanning the V3-V4 hypervariable region had better capture when compared to the primer pair for the V1-V3 region and was robust to spiking with human DNA. The pilot data generated using the V3-V4 region from the skin of healthy volunteers was consistent with these results, even at the genus level (Staphylococcus, Propionibacterium, Corynebacterium, Paracoccus, Micrococcus, Enhydrobacter and Deinococcus identified at similar abundances on both platforms)., Conclusions: The results suggest that the bacterial community diversity captured using the V3-V4 16S rRNA hypervariable region from sequencing using the MiSeq platform is comparable to the Roche454 GS Junior platform. These findings provide evidence that the optimised method can be used in human clinical samples of low bacterial biomass such as the investigation of the skin microbiota.

Introduction: Gene expression profiling is rapidly becoming a useful and informative tool in a much needed area of research. Identifying patients as to whether they will respond or not to a given treatment before prescription is not only essential to optimise treatment outcome but also to lessen the economic burden that such drugs can have on healthcare resources. In rheumatoid arthritis (RA), there is of yet no genetic/genomic biomarker which can accurately predict response to TNF inhibitor biologics prior to treatment, despite much interest in this area. Multiple studies have reported findings on potential candidate genes; however, due to relatively small sample sizes or lack of sufficient validation, results have been disappointingly inconsistent. The aim of this research was to further explore the predictive value of a previously reported association between CD11c expression and response to the TNF inhibitor biologics, adalimumab and etanercept., Methods: Real-time qPCR was performed using whole blood RNA samples obtained from seventy-five rheumatoid arthritis patients about to commence treatment with a TNF inhibitor biologic drug, whose response status was determined at 3-month follow-up using the EULAR classification criteria. Relative quantification of CD11c using the comparative CT method outputted differential expression between good-responders and non-responders as a fold-change., Results: Relative expression of CD11c in patients receiving TNF inhibitor biologics yielded a decrease of 1.025 fold in good-responders as compared to non-responders (p-value = 0.36). Upon stratification of patients dependent upon the specific drug administered, adalimumab or etanercept, similar findings to the full cohort were observed, decreases of 1.015 (p-value = 0.33) and 1.032 fold (p-value = 0.13) in good-responders compared to non-responders, respectively., Conclusion: The results from this study reveal that CD11c expression does not correlate with response to TNF inhibitor biologics when tested for within pre-treatment whole blood samples of rheumatoid arthritis patients.

Background: Genetic susceptibility to colonic inflammation is poorly defined at the gene level. Although Genome Wide Association studies (GWAS) have identified loci in the human genome which confer susceptibility to Inflammatory Bowel Disease (Crohn's and Ulcerative Colitis), it is not clear if precise loci exist which confer susceptibility to inflammation at specific locations within the gut e.g. small versus large intestine. Susceptibility loci for colitis in particular have been defined in the mouse, although specific candidate genes have not been identified to date. We have previously shown that infection with Trichuris muris (T. muris) induces chronic colitis in susceptible mouse strains with clinical, histological, and immunological homology to human colonic Crohn's disease. We performed an integrative analysis of colitis susceptibility, using an F2 inter-cross of resistant (BALB/c) and susceptible (AKR) mice following T. muris infection. Quantitative Trait Loci (QTL), polymorphic and expression data were analysed alongside in silico workflow analyses to discover novel candidate genes central to the development and biology of chronic colitis., Results: 7 autosomal QTL regions were associated with the establishment of chronic colitis following infection. 144 QTL genes had parental strain SNPs and significant gene expression changes in chronic colitis (expression fold-change ≥ +/-1.4). The T. muris QTL on chromosome 3 (Tm3) mapped to published QTL in 3 unrelated experimental models of colitis and contained 33 significantly transcribed polymorphic genes. Phenotypic pathway analysis, text mining and time-course qPCR replication highlighted several potential cis-QTL candidate genes in colitis susceptibility, including FcgR1, Ptpn22, RORc, and Vav3., Conclusion: Genetic susceptibility to induced colonic mucosal inflammation in the mouse is conserved at Tm3 and overlays Cdcs1.1. Genes central to the maintenance of intestinal homeostasis reside within this locus, implicating several candidates in susceptibility to colonic inflammation. Combined methodology incorporating genetic, transcriptional and pathway data allowed identification of biologically relevant candidate genes, with Vav3 newly implicated as a colitis susceptibility gene of functional relevance.

Introduction: In many European countries, restrictions exist around the prescription of anti-tumor necrosis factor (anti-TNF) treatments for rheumatoid arthritis (RA). Eligibility and response to treatment is assessed by using the disease activity score 28 (DAS28) algorithm, which incorporates one of two inflammatory markers, erythrocyte sedimentation rate (ESR) or C-reactive protein (CRP). Although DAS28-CRP provides a more reliable measure of disease activity, functional variants exist within the CRP gene that affect basal CRP production., Methods: DNA samples from the Biologics in Rheumatoid Arthritis Genetics and Genomics Study Syndicate (BRAGGSS) were genotyped for rs1205, rs1800947, and rs3091244 by using either TaqMan or the Sequenom MassARRAY iPLEX system., Results: Baseline CRP measurements were available for 599 samples with 442 also having data 6 months after treatment with an anti-TNF. For these 442 samples, the study had > 80% power to detect a clinically meaningful difference of 0.6 DAS28 Units for an allele frequency of 5%. Estimated haplotype frequencies corresponded with previous frequencies reported in the literature. Overall, no significant association was observed between any of the markers investigated and baseline CRP levels. Further, CRP haplotypes did not correlate with baseline CRP (P = 0.593), baseline DAS28-CRP (P = 0.540), or change in DAS28-CRP after treatment with an anti-TNF over a 6-month period (P = 0.302)., Conclusions: Although CRP genotype may influence baseline CRP levels, in patients with very active disease, no such association was found. This suggests that genetic variation at the CRP locus does not influence DAS28-CRP, which may continue to be used in determining eligibility for and response to anti-TNF treatment, without adjusting for CRP genotype.

Introduction: Juvenile idiopathic arthritis (JIA) is an umbrella term for all chronic childhood arthropathies and can be divided into seven subtypes. It includes the enthesitis related arthritis (ERA) subtype which displays symptoms similar to ankylosing spondylitis (AS) and juvenile-onset psoriatic arthritis which has similarities to psoriatic arthritis (PsA) and psoriasis (Ps). We, therefore, hypothesized that two well-established susceptibility loci for AS and Ps, ERAP1 and IL23R, could also confer susceptibility to these JIA subtypes., Methods: Single nucleotide polymorphisms (SNPs) in ERAP1 (rs30187) and IL23R (rs11209026) were genotyped in JIA cases (n = 1,054) and healthy controls (n = 5,200). Genotype frequencies were compared between all JIA cases and controls using the Cochrane-Armitage trend test implemented in PLINK. Stratified analysis by ILAR subtype was performed., Results: The ERA subtype showed strong association with ERAP1 SNP (P trend = 0.005). The IL23R SNP showed significant association in the PsA subtype (P trend = 0.04). The SNPs were not associated with JIA overall or with any other subtype., Conclusions: We present evidence for subtype specific association of the ERAP1 gene with ERA JIA and the IL23R gene with juvenile-onset PsA. The findings will require validation in independent JIA datasets. These results suggest distinct pathogenic pathways in these subtypes.

Introduction: Genome wide association studies, replicated by numerous well powered validation studies, have revealed a large number of loci likely to play a role in susceptibility to many multifactorial diseases. It is now well established that some of these loci are shared between diseases with similar aetiology. For example, a number of autoimmune diseases have been associated with variants in the PTPN22, TNFAIP3 and CTLA4 genes. Here we have attempted to define overlapping genetic variants between rheumatoid arthritis (RA), type 1 diabetes (T1D) and coeliac disease (CeD)., Methods: We selected eight SNPs previously identified as being associated with CeD and six T1D-associated SNPs for validation in a sample of 3,962 RA patients and 3,531 controls. Genotyping was performed using the Sequenom MassArray platform and comparison of genotype and allele frequencies between cases and controls was undertaken. A trend test P-value < 0.004 was regarded as significant., Results: We found statistically significant evidence for association of the TAGAP locus with RA (P = 5.0 × 10-4). A marker at one other locus, C1QTNF6, previously associated with T1D, showed nominal association with RA in the current study but did not remain statistically significant at the corrected threshold., Conclusions: In exploring the overlap between T1D, CeD and RA, there is strong evidence that variation within the TAGAP gene is associated with all three autoimmune diseases. Interestingly a number of loci appear to be specific to one of the three diseases currently studied suggesting that they may play a role in determining the particular autoimmune phenotype at presentation.

Association of a functional promoter polymorphism mapping to the Fc receptor-like 3 (FCRL3) gene has recently been reported and replicated with rheumatoid arthritis (RA) in Japanese populations. The aim of this study was to investigate association of the FCRL3 gene with RA in UK subjects. DNA was available from 1065 patients with RA and 2073 population controls from the UK. Four single nucleotide polymorphism (SNP) markers (FCRL3-169*C/T (fclr3&#95;3, rs7528684), fclr3&#95;4 (rs11264799), fclr3&#95;5 (rs945635), fclr3&#95;6 (rs3761959)) all previously associated with RA in a Japanese population were genotyped in 761 RA samples and 484 controls. In the remaining samples, only the putative disease causal polymorphism, FCRL3-169*C/T, was tested. Genotyping was performed using either the Sequenom MassArray iPlex platform or a 5' Allelic discrimination assay (Taqman, ABI). Extensive linkage disequilibrium was present across the promoter SNPs genotyped (r2 values = 0.60-0.98). Allele frequencies did not differ between RA cases and controls either for the putative disease causal polymorphism (odds ratio FCRL3-169*C allele = 0.97 (0.87-1.07), p = 0.51) or for the other SNPs tested. Similarly, no association was detected with RA using haplotype analysis or when stratification by shared epitope carriage or by presence of rheumatoid factor was undertaken. This study was powered to detect an effect size of 1.24 or greater for the FCRL3-169*C/T functional promoter polymorphism but no evidence for association was detected, suggesting that this gene will not have a substantial effect in determining susceptibility to RA in populations of Northern European descent.

Background: The selection of markers in association studies can be informed through the use of haplotype blocks. Recent reports have determined the genomic architecture of chromosomal segments through different haplotype block definitions based on linkage disequilibrium (LD) measures or haplotype diversity criteria. The relative applicability of distinct block definitions to association studies, however, remains unclear. We compared different block definitions in 6.1 Mb of chromosome 17q in 189 unrelated healthy individuals. Using 137 single nucleotide polymorphisms (SNPs), at a median spacing of 15.5 kb, we constructed haplotype block maps using published methods and additional methods we have developed. Haplotype tagging SNPs (htSNPs) were identified for each map., Results: Blocks were found to be shorter and coverage of the region limited with methods based on LD measures, compared to the method based on haplotype diversity. Although the distribution of blocks was highly variable, the number of SNPs that needed to be typed in order to capture the maximum number of haplotypes was consistent., Conclusion: For the marker spacing used in this study, choice of block definition is not important when used as an initial screen of the region to identify htSNPs. However, choice of block definition has consequences for the downstream interpretation of association study results.